Methods and apparatus for template capture and normalization for submicroliter reaction

• US 20030032052A1
• Filed: 09/30/2002
• Published: 02/13/2003
• Est. Priority Date: 08/02/1999
• Status: Active Grant

• Current Assignee: IntegenX Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Dynamics Inc. (General Electric Company)
• Inventors: Jovanovich, Stevan, Hadd, Andy
• Granted Patent: US 6,927,045 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00286 Reactor vessels with top and bottom openings
• CPC Class Codes: B01J 2219/00369 in multiple or parallel arrangements
• CPC Class Codes: B01J 2219/00511 Walls of reactor vessels
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00691 using robots
• CPC Class Codes: B01J 2219/00702 Processes involving means for analysing and characterising the products
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2300/1844 using fans
• CPC Class Codes: B01L 2400/0409 centrifugal forces
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 7/52 with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples amplification or hybridisation processes per se C12Q1/68; controlling sequential reactions for synthesis B01J19/0046
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: Y10S 435/809 : Incubators or racks or holders for culture plates or containers
• CPC Class Codes: Y10T 436/113332 : with conveyance of sample along a test line in a container or rack
• CPC Class Codes: Y10T 436/143333 : Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/2575 : Volumetric liquid transfer

Abstract

Methods for preparing nanoscale reactions using nucleic acids are presented. Nucleic acids are captured saturably, yet reversibly, on the internal surface of the reaction chamber, typically a capillary. Excess nucleic acid is removed and the reaction is performed directly within the capillary. Alternatively, the saturably bound nucleic acid is eluted, dispensing a metered amount of nucleic acid for subsequent reaction in a separate chamber. Devices for effecting the methods of the invention and a system designed advantageously to utilize the methods for high throughput nucleic acid sequencing reactions are also provided.

Claims

1. A method of performing a DNA sequencing reaction, comprising:

immobilizing template DNA directly on a substrate, and then contacting said template DNA with a reaction mixture that effects said DNA sequencing reaction.

2. The method of claim 1 wherein said template DNA is double stranded DNA.

3. The method of claim 1 wherein said template DNA is single stranded DNA.

4. The method of claim 2 or 3 wherein said template DNA is prepared according to the polymerase chain reaction.

5. The method of claim 1 wherein said template DNA is immobilized by:

contacting the substrate with a solution of the template DNA for time sufficient for the DNA to become immobilized.

6. The method of claim 1 further comprising removing the solution, wherein said removing step occurs after said immobilizing step.

7. The method of claim 6 further comprising washing the substrate, wherein said washing step occurs after said removing step.

8. The method of claim 7 wherein said washing removes impurities.

9. The method of claim 7 wherein said washing is effected using an ethanol solution of at least about 70% ethanol.

10. The method of claim 7 further comprising drying the substrate, wherein said drying step occurs after said washing step.

11. The method of claim 6 further comprising eluting the DNA from the substrate, wherein said elution step occurs after said removing step.

12. The method of claim 7 further comprising subjecting the template DNA in contact with the reaction mixture to at least one thermal cycle.

13. The method of claim 12 further comprising dispensing the products of the DNA sequencing reaction, wherein said dispensing step occurs after the reaction has been effected.

14. The method of claim 1 or 5 wherein said template DNA immobilization is effected by saturable binding to said substrate.

15. The method of claim 5 wherein said DNA template solution comprises a substance that effects the immobilization of the template DNA directly on the substrate by a noncovalent interaction.

16. The method of claim 15 wherein said noncovalent interaction is an electrostatic interaction.

17. The method of claim 15 wherein said substance is a chaotropic agent.

18. The method of claim 17 wherein said chaotropic agent is selected from the group consisting of:

urea, sodium perchlorate, potassium perchlorate, sodium bromide, potassium bromide, sodium iodide, potassium iodide, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, sodium isothiocyanate, potassium isothiocyanate, guanidine hydrochloride, guanidine isothiocyanate, lithium chloride, sodium trichloroacetate, dimethylsulfoxide, tetra-amine halides, tetraethylamine chloride, and potassium trichloroacetate.

19. The method of claim 1 wherein said reagent mixture comprises an oligonucleotide primer, a DNA polymerase, and deoxynucleotide triphosphates (dNTPs).

20. The method of claim 19 wherein said reagent mixture elutes the DNA from the substrate.

21. The method of claim 19 wherein said reagent mixture further comprises a limiting concentration of dideoxynucleotide triphosphates.

22. The method of claim 21 wherein each of said dideoxynucleotide triphosphates is conjugated to a fluorophore.

23. The method of claim 19 wherein said primer is complementary to a plurality of continuously arranged nucleotides in at least one strand of the DNA template.

24. The method of claim 23 wherein said primer is conjugated to a fluorophore.

25. The method of claim 24 wherein said fluorophore is an energy transfer fluorophore.

26. The method of claim 19 wherein said DNA polymerase is thermostable.

27. The method of claim 19 further comprising subjecting the template DNA in contact with the reaction mixture to at least one thermal cycle.

28. The method of claim 1 wherein said substrate is at least one internal surface of an enclosed channel.

29. The method of claim 28 further comprising filling said enclosed channel with a fluid.

30. The method of claim 28 further comprising sealing the enclosed channel against egress by fluids contained therein.

31. The method of claim 28, wherein said substrate is glass.

32. The method of claim 28, wherein said substrate is a metal.

33. The method of claim 28, wherein said substrate is a metalloid.

34. The method of claim 28 wherein said enclosed channel is formed by a capillary tube.

35. The method of claim 28 wherein said enclosed channel is formed by a depression upon at least part of which is disposed a cover, wherein said cover encloses said channel.

36. A product of a DNA sequencing reaction effected by the method of any one of claims 1, 5, 6, 7, 11, 12, 13, 15, 19, or 28.

37. A DNA sequence derived from the product of said DNA sequencing reaction of claim 36.

38. The DNA sequence of claim 37 embodied in a computer readable medium.

39. A computer data signal embodied in a carrier wave, wherein the computer data signal comprises the DNA sequence of claim 37.

82. The substrate of any one of claims 1, 5, 6, 7, 10, 15, 16, 17, 18, 28, 34, 35, 40, 41, 42, or 50 on which said nucleic acid or said DNA is immobilized.

40. A method of obtaining a predetermined approximate mass of a nucleic acid, comprising:

immobilizing said nucleic acid directly on a substrate, wherein said substrate saturably binds said nucleic acid.

41. The method of claim 40 wherein said nucleic acid is RNA.

42. The method of claim 40 wherein said nucleic acid is DNA.

43. The method of claim 42 wherein said DNA is double stranded DNA.

44. The method of claim 42 wherein said DNA is single stranded DNA.

45. The method of claim 42 wherein said DNA is produced by polymerase chain reaction.

46. The method of claim 42 wherein said DNA is isolated from eukaryotic cells, prokaryotic cells, archaea cells, viruses, or bacteriophage.

47. The method of claim 42 wherein said DNA is produced according to a chemical synthetic process.

48. The method of claim 40 wherein said nucleic acid is used in an enzymatic reaction.

49. The method of claim 42 wherein said DNA is used in an enzymatic reaction

50. The method of claim 49 wherein said reaction is a DNA sequencing reaction.

61. The method of claim 40 or 51 wherein the nucleic acid is immobilized by:

contacting the substrate with a solution of the nucleic acid for time sufficient for the nucleic acid to become immobilized.

62. The method of claim 61 wherein said nucleic acid solution comprises a substance that effects the immobilization of the nucleic acid directly on the substrate by a noncovalent interaction.

63. The method of claim 62 wherein said noncovalent interaction is an electrostatic interaction.

64. The method of claim 62 wherein said substance is a chaotropic agent.

65. The method of claim 64 wherein said chaotropic agent is selected from the group consisting of:

urea, sodium perchlorate, potassium perchlorate, sodium bromide, potassium bromide, sodium iodide, potassium iodide, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, sodium isothiocyanate, potassium isothiocyanate, guanidine hydrochloride, guanidine isothiocyanate, lithium chloride, sodium trichloroacetate, dimethylsulfoxide, tetra-amine halides, tetraethylamine chloride, and potassium trichloroacetate.

66. The method of claim 61 further comprising removing the solution, wherein said removing step occurs after said immobilizing step.

67. The method of claim 66 further comprising washing the substrate, wherein said washing step occurs after said removing step.

68. The method of claim 67 further comprising drying the substrate, wherein said drying step occurs after said washing step.

69. The method of either of claims 40 or 51 wherein said substrate is at least one wall of an enclosed channel.

70. The method of claim 69 further comprising filling said enclosed channel with a fluid.

71. The method of claim 69, wherein said substrate is glass.

72. The method of claim 69, wherein said substrate is a metal.

73. The method of claim 69, wherein said substrate is a metalloid.

74. The method of claim 69 wherein said enclosed channel is formed by a capillary tube.

75. The method of claim 69 wherein said enclosed channel is formed by a depression upon at least part of which is disposed a cover, wherein said cover creates a septum preventing fluid communication.

51. A method of dispensing a predetermined approximate mass of a nucleic acid, comprising:

immobilizing said nucleic acid directly on a first substrate, wherein said substrate saturably binds said nucleic acid, and

transferring said nucleic acid to a second substrate.

52. The method of claim 51 further comprising eluting the nucleic acid from said substrate.

53. The method of claim 51 wherein said nucleic acid is RNA.

54. The method of claim 51 wherein said nucleic acid is DNA.

55. The method of claim 54 wherein said DNA is double stranded DNA.

56. The method of claim 54 wherein said DNA is single stranded DNA.

57. The method of claim 51 wherein said nucleic acid is transferred to said second substrate in a fluid volume of about 1-1000 nanoliters.

58. The method of claim 51 wherein said nucleic acid is transferred to said second substrate in a fluid volume of about 1-500 nanoliters.

59. The method of claim 51 wherein said nucleic acid is transferred to said second substrate in a fluid volume of about 1-100 nanoliters.

60. The method of claim 51 wherein said nucleic acid is transferred to said second substrate in a fluid volume of about 1-10 nanoliters.

76. A method for obtaining a predetermined approximate mass of size-selected DNA, comprising:

size-selecting DNA to be normalized, and immobilizing said size-selected DNA directly on a substrate,

wherein said substrate saturably binds said nucleic acid.

77. The method of 76 wherein said DNA to be obtained is plasmid or episomal DNA.

78. The method of 77 wherein said size-selection is effected by the exclusion of genomic DNA from the bore of an enclosed channel.

79. The method of 78 wherein said enclosed channel is a capillary tube.

80. The method of 78 or 79 wherein said immobilization is effected by contacting at least one wall of said enclosed channel or capillary tube with a solution comprising DNA and a chaotropic agent.

81. The method of claim 80 wherein said chaotropic agent is selected from the group consisting of:

urea, sodium perchlorate, potassium perchlorate, sodium bromide, potassium bromide, sodium iodide, potassium iodide, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, sodium isothiocyanate, potassium isothiocyanate, guanidine hydrochloride, guanidine isothiocyanate, lithium chloride, sodium trichloroacetate, dimethylsulfoxide, tetra-amine halides, tetraethylamine chloride, and potassium trichloroacetate.

83. A method of introducing into an enzymatic reaction a predetermined approximate mass of nucleic acid, comprising:

saturably capturing, directly onto an interior surface of a chamber in which said enzymatic reaction is to be performed, a predetermined approximate mass of nucleic acid from an excess thereof, and then

removing said excess.

84. The method of claim 83 wherein an enzymatic reaction mixture is introduced into said reaction chamber after the excess mass of nucleic acid is removed.

85. The method of claim 84 wherein said reaction mixture comprises an enzyme selected from among the group consisting of:

polymerase, DNA polymerase, Sequenase™

, Thermo Sequenase II™

, RNA polymerase, reverse transcriptase, thermostable polymerase, ligase, kinase, phosphatase, restriction enzyme, endonuclease, exonuclease, topoisomerase, terminal transferase, glycosidase, and methylase.

86. A method of verifying the sequence of template DNA in solution, wherein said solution has been, or is desired to be contacted to a first substrate as part of a spatially addressable array, comprising:

immobilizing the template DNA directly on a second substrate, wherein said template DNA is immobilized by;

contacting the second substrate with the solution of template DNA for time sufficient for the DNA to become immobilized, and then

contacting said template DNA with a reaction mixture that effects said DNA sequencing reaction, wherein the composition of the template DNA solution to be contacted to said first and second substrates is essentially identical.